Evaluation of the impact of vindoline, an active components of Catharanthus roseus, on rat hepatic cytochrome P450 enzymes by using a cocktail of probe drugs

The objection of this study was to investigate the effects of vindoline(VDL) on the cytochrome P450 (CYP 450) isoforms (CYP1A2, 2B, 2C11, 2D1 and 3A) in rats. Firstly, the rats were randomly divided into VDL pretreatment group and blank group, each group had six rats. VDL pretreatment group was administrated VDL (20 mg·kg-1) by oral gavage for fifteen days consecutively, and the equivalent CMC-Na solution without VDL was given to the blank group by gavage. Secondly, a cocktail of caffeine, bupropion, diclofenac, dextromethorphan and midazolam was then administered on the sixteenth day. Finally, blood samples were collected at the specified time point, and the plasma concentration of the probe drug was determined by UHPLC-QTOF-MS/MS. The effects of VDL on the activity of these CYP enzymes in rats were evaluated by pharmacokinetic parameters. VDL pretreatment group compared with the blank group, accelerated the metabolism of diclofenac, and weakened the metabolism of caffeine. These results suggested that VDL could induce the activity of CYP2C11, and inhibits the activity of CYP1A2, but had no significant effects on CYP2B, CYP2D1 and CYP3A. The results in this study can provide beneficial information for the later clinical application of VDL.


Introduction
Catharanthus roseus (L.) G. Don (C. roseus), an aboriginal perennial plant species of Madagascar, is often known as the Madagascar periwinkle [1,2]. C. roseus is a tropical perennial subshrub known to produce about 130 alkaloids, such as vinblastine, vincristine, vinleurosine, vindoline, and catharanthine [3,4]. In some countries, leaf and whole plant decoctions are used as home remedies for diabetes [5,6]. Previous literature shows that the C. roseus leaf juice has significant antidiabetes activity, which may help prevent diabetes complications and play a good auxiliary role in the current antidiabetes drugs [5,7]. Vindoline (VDL) is a semi-synthetic intermediate vinca alkaloid, which can be extracted from C roseus, it is reported to have a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 anti diabetic properties in diabetes induced animal models [8,9]. Recently, it was reported that VDL has the inhibitory effect on protein tyrosine phosphatase 1B (PTP-1B), so it can be used as an "insulin sensitizer" in the treatment of type 2 diabetes mellitus (T2DM) [10][11][12].
Diabetes is a lifelong metabolic diseases characterized by chronic hyperglycemia due to multiple etiologies [13]. According to the World Health Organization (WHO), diabetes has up to 100 complications, and which is currently one of the most known complications [14]. More than half of the deaths from diabetes were due to cardio cerebrovascular causes, and 10% were due to renal lesions. Clinical data shows that 30% -40% of patients with diabetes will have at least one complication about 10 years after the onset of diabetes [15]. So diabetic patients usually take hypoglycemic drugs for a long time and even suffer from lifelong drug treatment, and it is often in combination with other drugs [16]. Taking multiple drugs at the same time can easily create drug-drug interactions (DDIs). As a potential hypoglycemic agent, VDL may be used in combination with other drugs. Therefore, it is necessary to clarify the effects of VDL on CYP enzymes activity [17].
The cytochrome P450 (CYP450) enzyme system has the greatest relationship with drug metabolism [18]. It mainly exists in human liver, and also has a small amount of expression in kidney, small intestine, lung and brain [19]. It participates in the biological transformation of many endogenous and exogenous substances (including most clinical drugs) of organisms [20]. DDIs should be considered in clinical use, mainly because there are many drugs that can enhance or weaken the activity of CYP (inducer), change the rate of drug metabolism, and affect the effect of drugs [21].
Recently a quadrupole time-of-flight mass (TripleTOF-MS) spectrometer has been routinely used as an effective and reliable analytical instrument [22]. The TripleTOF 5600+ system has a new ultra-high sensitivity designs, it can achieve high resolution and open a very narrow detection window without decreasing the sensitivity. At this time, it eliminates the interference of various impurities and has high selectivity. Therefore, the quantitative sensitivity can be comparable to the MRM sensitivity of API 4000 or API 5000; at the same time, the scanning speed as high as 100 spectra/s ensures that sufficient data points can be collected even for the peak width of UHPLC in a few seconds, so as to obtain excellent reproducibility and reliability.
Theoretically, human CYP1A2 is homolog to rat CYP1A2, human CYP2B is homolog to rat CYP2B, human CYP2C9 is homolog to rat CYP2C11, human CYP2D6 is homolog to rat CYP2D1 and human CYP3A4 is homolog to rat CYP3A [20,23]. So rats were chosen as experimental animal in this research. To investigate the effects of VDL on rat hepatic CYP enzymes, this study adopted a probe cocktail approach using caffeine (CYP1A2), bupropion (CYP2B), dextromethorphan (CYP2C11), diclofenac acid (CYP2D1) and midazolam (CYP3A). Meanwhile, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS) method was used to detect these five probe drugs, which can provide a highly selective and sensitive assay with low limits of quantification.

Instrumentations
Samples were analyzed by UHPLC-QTOF-MS/MS, which consisted of a Prominence TM UHPLC System (Shimadzu, Japan) and a Triple TOF TM 5600+ system (AB SCIEX, USA) equipped with Duo-Spray TM ion sources in the electrospray ionization (ESI) technology.

Mass spectrometric conditions
The mass detection was accomplished in multiple reaction monitoring (MRM HR ) mode. Analyst 1 TF 1.7 software (AB SCIEX, USA) and MultiQuant 3.0.1 (AB SCIEX, USA) were used for instrument control, data acquisition as well as data processing. The ESI source was set to positive ion mode with ionization conditions as follows: ion spray voltage floating, (+) 5.5 kV; ion source heater, 550˚C; curtain gas, 25 psi; ion source gas 1, 55 psi; and ion source gas 2, 55 psi. The detected ions and collision energy (CE) of the five cocktail probe drugs are shown in Table 1. The collision energy expansion (CES) is 0 eV. A narrow extraction window (±0.02 Da) was used in MultiQuant 3.0.1 to leverage the high resolution data.

Preparation of calibration curve samples and quality control (QC) samples.
Five probe drug stock solutions with a concentration of 1mg/mL were prepared in methanol. A series of working solutions were obtained by diluting the stock solution with methanol. Additionally, the phenacetin (IS) working solution was obtained by diluting the stock standard solution with methanol to yield the final concentration of 0.2 μg/mL. All the solutions were stored at 4˚C until analysis.
Add the working solution into 100 μL blank plasma to prepare the calibration standards and quality control (QC) samples.  10 μL of methanol and 10 μL of IS working solution were added into 100 μL of rat plasma sample. Then adding 300 acetonitrile to precipitate protein, the mixture was vortexed for 5min, and centrifuged at 15000 rpm for 10min, the supernatant was transferred to another tube, evaporated to dryness under a stream of nitrogen. The residue was reconstituted with 150 μL of 80% acetonitrile and centrifuged at 15000 rpm for 10 min. Before injection, 2 μL of supernatant was used for UHPLC-QTOF-MS/MS analysis.

Application of the analytical method in CYP450 activity study
Male Sprague-Dawley rats (Certificate No.SCXK 2013-1-003; weighting 250±20g; 7-8 weeks of age) were supplied by the Experimental Animal Center of Hebei Laboratory Animal Center (Shijiazhuang, China). Before the experiment, rats were fed in a breeding room with a temperature of 22-25˚C, a relative humidity of 55-60% and a 12h light/dark cycle for a week. During this period, rats ate and drank normally, and fasted overnight before administration. All experiments on rats were conducted in accordance with the guidelines of the Committee on the Care and Use of Laboratory Animals in our laboratory.

Statistical analysis
The pharmacokinetic parameters of the five probe drugs were derived with a non-linearre gression iterative program, and calculated with the DAS 3.0 pharmacokinetic program (Chinese Pharmacological Society, Beijing, China). The statistical analysis used SPSS 21.0 (IBM SPSS, USA), and compared the pharmacokinetic characteristics of five probe drugs with Oneway analysis of variance (ANOVA). When P<0.05, the difference was considered statistically significant.

Results
An efficient and reliable UHPLC-MS method for simultaneous determination of caffeine, bupropion, diclofenac, dextromethorphan, and midazolam is established, thus can be applied to activity evaluation of CYP1A2, 2B, 2C11, 2D1 and 3A with a cocktail approach.

Method validation
The chromatograms of the blank plasma samples, blank plasma samples spiked with analytes at LLOQ and plasma samples from rats 1h after oral administration of cocktail solution are shown in Fig 1. The calibration curves were obtained by using a weighting factor of 1/x 2 . The calibration curves, linear ranges, correlation coefficients and LLOQs of the five cocktail probe drugs are shown in Table 2. The real values of each calibration concentration were between 89% and 112% of the theoretical values. None of the RE and RSD values of the intra-day and inter-day accuracy and precision exceeded 15%.

Effects of VDL on CYP450 activities in rats
The concentration-time curves and pharmacokinetic parameters are shown in Figs 2-6 and Tables 3-7, respectively.

Cocktail probe drugs method
The cocktail probe drugs method is a screening tool to predict the enzyme activity. In 1990, Breimer, DD et al. first used multiple probe substrates simultaneously to evaluate the activity of multiple CYP450 subtypes of enzymes, which called "Cocktail probe drug approach". This approach is capable of measurement the activity of multiple CYP enzymes after the administration of multiple CYP-specific substrates in a single experiment.
The V max and K m values of cocktail probe drug approach and single probe approach are consistent, thus cocktail probe drug approach can replace single probe approach for the evaluation of CYP450 activity [25]. So far, the cocktail method can allow fast routine measurement of the multiple CYP enzyme activities in a single experiment, and this method also can minimize the confounding influence of inter-individual and intra-individual variability [21,26,27]. Therefore, the cocktail method is a powerful tool for comprehensively evaluating drug interactions involving multiple CYP enzymes simultaneously.
According to the "Drug Interaction Research" guidelines released by the FDA in the United States [28], the selection of in vivo study probe drugs depends on the CYP450 enzyme. Generally, probe drugs that specifically bind to isoenzymes should be selected. Therefore, according to the recommended guidelines, we have chosen the following probe drugs: (1) Midazolam (CYP3A); (2) Caffeine (CYP1A2); (3) Bupropion (CYP2B); (4) Diclofenac (CYP2C9); (5) Dextromethorphan (CYP2D6). In the experiment, the selection of probe drug dosage is also very important. The dose range of the probe drug was determined through literature [29][30][31], then preliminary experiments were conducted to determine the actual dosage, ensuring that low doses of composite probes were administered to experimental animals within the allowable range of detection capabilities, which can reduce the possibility of potential drug-drug interactions between probe drugs [21]. Finally, we determine the dose of the probe drugs, which is

Induction and inhibition of CYP450 isoform activity by VDL
In the treatment of diabetes, patients usually need to take multiple drugs, and the combination of multiple drugs is very likely to cause DDIs [32]. If several drugs are metabolized through the same enzyme, the drug efficacy will increase or decrease, which will lead to treatment failure [33]. As a potential anti diabetes drug, it is necessary to study the effect of VDL on enzyme activity. In this study, we found that VDL tended to be the inducer of CYP2C11 (CYP2C9 in human). Stephen S. et al. reported that the nuclear receptor CAR (Constitutive androgen receptor) as an effective regulator of CYP2C9 transcription at both the mRNA level and in promoter assays [34]. So we think that VDL might upregulate the transcription level of CYP2C9 through the CAR receptor. In addition, we found that VDL had an inhibitive effect on     CYP1A2. It is reported that nitrogen-containing heterocyclic compounds can bind tightly to the prosthetic haem iron of CYP proteins; this binding often exhibits reversible inhibition on CYP enzymes [35]. VDL is a single indole alkaloid, which have nitrogen-containing pyrrole rings. So we think that the inhibitory effect of VDL on CYP1A2 may be related to the nitrogen-containing heterocycles in VDL.
The induction of CYP leads to the increase of the synthesis or activity of CYP, thus accelerating the metabolism of drugs eliminated by this procedure, leading to the reduction of plasma drug concentration and treatment delay [36]. In this study, treatment of rats with VDL decreased the t 1/2 , C max , AUC 0-t , AUC 0-1 and increased clearance (CL) of diclofenac compared with those in the blank control. It is suggested that VDL could induce CYP2C11, which is homolog to human CYP2C9. CYP2C9 is one of the most important metabolic enzymes in the CYP enzyme family. About 15% of clinical drugs are metabolized by CYP2C9, including celecoxib, phenytoin sodium, diazepam, tolbutamide, diclofenac acid, losartan and S-warfarin [37,38]. In clinic, when applied in combination, VDL may interact with all drugs metabolized by CYP2C9, especially for drugs with a narrow therapeutic window, such as phenytoin sodium. Therefore, VDL should be avoided in combination with these drugs.
According to the experimental results, the activity of CYP1A2 might be inhibited by VDL. Enzyme inhibition refers to those drugs can inhibit the activity of enzymes during the combined use of drugs, resulting in slower metabolism of the combined drugs, making the concentration of drugs in the body higher than the normal concentration when used alone, and enhancing the effect [35]. For drugs with narrow treatment window, serious adverse reactions may occur [39]. CYP1A2 accounts for only 13% in the CYP enzyme family, but many important marketed drugs are metabolized by CYP1A2 [40]. In the future clinical application of VDL, it is likely to be combined with many drugs, including antihypertensive drugs, hypoglycemic drugs, and lipid-lowering drugs and so on [41], if the main metabolic enzyme of these drugs is CYP1A2, because VDL has an inhibitory effect on CYP1A2, the plasma concentration of these drugs will increase compared with the concentration when they are used alone, which can cause adverse effects, and may endanger life in serious cases [42,43]

Analysis of in vitro and in vivo experimental results
In the previous in vitro experiments, it was found that VDL could inhibit CYP2D1 activity, and had weak inhibitory effect on CYP2C11 and CYP3A (S1 Fig), which was inconsistent with the results of in vivo experiments [44]. The reasons for this phenomenon may be as follows: (1) Influence of in vivo and in vitro action time and dosage: in vitro experiments, VDL and probe drugs were directly incubated with CYP450 enzymes in rat liver microsome, while in vivo experiments, rats were pretreated with VDL for 15 days, and then the probe substrate was administered on the 16 days. The expression environment of the enzyme, and the dosage in rat are differences, it may cause VDL to induce or inhibit the CYP450 enzyme in different ways.
(2) Compared with in vivo experiment, the environment of the enzymatic reaction was artificially simulated in vitro, and the interference of other complex factors in the body was excluded. But in vivo experiments reflect the metabolism of drugs in the physiological state of the whole living organism. This difference of in vitro and in vivo environment may cause changes in the activity and quantity of CYP450 enzymes, which may lead to inconsistencies in the results of in vitro and in vivo experiments [45]. This study suggested that examining the effects of drugs on CYP450 enzyme activity, in vitro and in vivo experiments are equally important. Preliminary exploration of in vitro experiments can provide ideas for further in vivo experiments, but whether the in vitro experimental data can be used to directly predict the interaction results in vivo remains to be further verified.

Conclusion
In this study, the in vivo results was showed that VDL inhibited the activity of CYP1A2, and induced the activity of CYP2C9, but had no effects on the activities of CYP2B, CYP2D6 and CYP3A4. Based on the above findings, we need follow with interest potential DDIs regulated by CYP1A2 and 2C9, when VDL combined with other drugs (particularly for drugs with narrow therapeutic window). However, the relevant research and clinical significance of human still need further study.